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Local and global analysis of endocytic patch dynamics in fission yeast using a new "temporal superresolution" realignment method.

Berro J, Pollard TD - Mol. Biol. Cell (2014)

Bottom Line: These methods allowed us to extract new information about endocytic actin patches in wild-type cells from measurements of the fluorescence of fimbrin-mEGFP.We show that the time course of actin assembly and disassembly varies <600 ms between patches.Our methods also show that the number of patches in fission yeast is proportional to cell length and that the variability in the repartition of patches between the tips of interphase cells has been underestimated.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology Department of Molecular Biophysics and Biochemistry Nanobiology Institute, Yale University, New Haven, CT 06520-8103 Institut Camille Jordan, UMR CNRS 5208, Université de Lyon, 69622 Villeurbanne-Cedex, France Centre de Génétique et de Physiologie Moléculaire et Cellulaire, UMR CNRS 5534, Université de Lyon, 69622 Villeurbanne-Cedex, France.

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The time course of fimbrin and capping protein appearance and disappearance in endocytic patches is highly reproducible. (A) Example of three patches tracked with fimbrin (Fim1p-mEGFP) in wild-type cells (1 patch per line with the color code for each patch on the right). Each image represents the sum of Fim1p-mEGFP fluorescence intensities from five confocal sections at 1-s intervals. The horizontal gray lines represent the approximate position of the plasma membrane, and the vertical gray ticks mark the horizontal positions of each patch in the first image. Fim1p-mEGFP fluorescence intensity is color-coded from low to high intensities: black–blue–orange–red–yellow–white (ImageJ “fire” lookup table). Colored scale bars: 500 nm. (B) The variability in timing between patches is less than the measurement interval. Time course of the fluorescence of fimbrin-mEGFP in 24 patches matched by continuous alignment and normalized to their peak values. Each dot corresponds to a time point of a given track. Each color corresponds to a different track. The black curve is the average time for the normalized fluorescence to reach a given value. The horizontal black lines are the SDs of these mean times and are plotted in Figure S3A. The gray curves represent the average ± 1 SD. (C) Average of two-color data sets realigned using the data from only one channel. Blue, Acp1p-mEGFP, and red, Fim1p-mCherry: realigned using only Fim1p-mCherry data; teal, Acp1p-mEGFP, and purple, Fim1p-mCherry: realigned using only Acp1p-mEGFP data. The raw data used for this alignment are the same as for Figure S1K in Berro and Pollard (2014); N = 19. (D) Numbers of fimbrin molecules in 3 endocytic patches from A vs. time. These and 21 other data sets were aligned on the same timescale by temporal superresolution alignment of the intensities to calculate the averaged numbers over time (black curve) ± 1 SD from the average (gray curves). Time zero is the time when the average number of fimbrin molecules peaked.
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Figure 3: The time course of fimbrin and capping protein appearance and disappearance in endocytic patches is highly reproducible. (A) Example of three patches tracked with fimbrin (Fim1p-mEGFP) in wild-type cells (1 patch per line with the color code for each patch on the right). Each image represents the sum of Fim1p-mEGFP fluorescence intensities from five confocal sections at 1-s intervals. The horizontal gray lines represent the approximate position of the plasma membrane, and the vertical gray ticks mark the horizontal positions of each patch in the first image. Fim1p-mEGFP fluorescence intensity is color-coded from low to high intensities: black–blue–orange–red–yellow–white (ImageJ “fire” lookup table). Colored scale bars: 500 nm. (B) The variability in timing between patches is less than the measurement interval. Time course of the fluorescence of fimbrin-mEGFP in 24 patches matched by continuous alignment and normalized to their peak values. Each dot corresponds to a time point of a given track. Each color corresponds to a different track. The black curve is the average time for the normalized fluorescence to reach a given value. The horizontal black lines are the SDs of these mean times and are plotted in Figure S3A. The gray curves represent the average ± 1 SD. (C) Average of two-color data sets realigned using the data from only one channel. Blue, Acp1p-mEGFP, and red, Fim1p-mCherry: realigned using only Fim1p-mCherry data; teal, Acp1p-mEGFP, and purple, Fim1p-mCherry: realigned using only Acp1p-mEGFP data. The raw data used for this alignment are the same as for Figure S1K in Berro and Pollard (2014); N = 19. (D) Numbers of fimbrin molecules in 3 endocytic patches from A vs. time. These and 21 other data sets were aligned on the same timescale by temporal superresolution alignment of the intensities to calculate the averaged numbers over time (black curve) ± 1 SD from the average (gray curves). Time zero is the time when the average number of fimbrin molecules peaked.

Mentions: We collected data on the time course of the accumulation and disappearance of the actin cross-linking protein fimbrin in endocytic actin patches to test our alignment method on a dynamic process in live cells. We imaged fission yeast cells expressing Fim1p-mEGFP at 25°C in EMM5S medium on five consecutive confocal sections spaced at 360 nm along the z-axis. These imaging conditions allowed us to track patches during most of their lifetimes and verify that their entire fluorescence signal was collected and did not overlap with other patches. We restricted our analysis to patches with fluorescence in only three consecutive slices, which we believe to be single isolated patches. We used five successive rounds of continuous alignment to align our data on the time course of the numbers of fimbrin molecules in well-separated actin patches (Figure 3, B and C).


Local and global analysis of endocytic patch dynamics in fission yeast using a new "temporal superresolution" realignment method.

Berro J, Pollard TD - Mol. Biol. Cell (2014)

The time course of fimbrin and capping protein appearance and disappearance in endocytic patches is highly reproducible. (A) Example of three patches tracked with fimbrin (Fim1p-mEGFP) in wild-type cells (1 patch per line with the color code for each patch on the right). Each image represents the sum of Fim1p-mEGFP fluorescence intensities from five confocal sections at 1-s intervals. The horizontal gray lines represent the approximate position of the plasma membrane, and the vertical gray ticks mark the horizontal positions of each patch in the first image. Fim1p-mEGFP fluorescence intensity is color-coded from low to high intensities: black–blue–orange–red–yellow–white (ImageJ “fire” lookup table). Colored scale bars: 500 nm. (B) The variability in timing between patches is less than the measurement interval. Time course of the fluorescence of fimbrin-mEGFP in 24 patches matched by continuous alignment and normalized to their peak values. Each dot corresponds to a time point of a given track. Each color corresponds to a different track. The black curve is the average time for the normalized fluorescence to reach a given value. The horizontal black lines are the SDs of these mean times and are plotted in Figure S3A. The gray curves represent the average ± 1 SD. (C) Average of two-color data sets realigned using the data from only one channel. Blue, Acp1p-mEGFP, and red, Fim1p-mCherry: realigned using only Fim1p-mCherry data; teal, Acp1p-mEGFP, and purple, Fim1p-mCherry: realigned using only Acp1p-mEGFP data. The raw data used for this alignment are the same as for Figure S1K in Berro and Pollard (2014); N = 19. (D) Numbers of fimbrin molecules in 3 endocytic patches from A vs. time. These and 21 other data sets were aligned on the same timescale by temporal superresolution alignment of the intensities to calculate the averaged numbers over time (black curve) ± 1 SD from the average (gray curves). Time zero is the time when the average number of fimbrin molecules peaked.
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Related In: Results  -  Collection

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Figure 3: The time course of fimbrin and capping protein appearance and disappearance in endocytic patches is highly reproducible. (A) Example of three patches tracked with fimbrin (Fim1p-mEGFP) in wild-type cells (1 patch per line with the color code for each patch on the right). Each image represents the sum of Fim1p-mEGFP fluorescence intensities from five confocal sections at 1-s intervals. The horizontal gray lines represent the approximate position of the plasma membrane, and the vertical gray ticks mark the horizontal positions of each patch in the first image. Fim1p-mEGFP fluorescence intensity is color-coded from low to high intensities: black–blue–orange–red–yellow–white (ImageJ “fire” lookup table). Colored scale bars: 500 nm. (B) The variability in timing between patches is less than the measurement interval. Time course of the fluorescence of fimbrin-mEGFP in 24 patches matched by continuous alignment and normalized to their peak values. Each dot corresponds to a time point of a given track. Each color corresponds to a different track. The black curve is the average time for the normalized fluorescence to reach a given value. The horizontal black lines are the SDs of these mean times and are plotted in Figure S3A. The gray curves represent the average ± 1 SD. (C) Average of two-color data sets realigned using the data from only one channel. Blue, Acp1p-mEGFP, and red, Fim1p-mCherry: realigned using only Fim1p-mCherry data; teal, Acp1p-mEGFP, and purple, Fim1p-mCherry: realigned using only Acp1p-mEGFP data. The raw data used for this alignment are the same as for Figure S1K in Berro and Pollard (2014); N = 19. (D) Numbers of fimbrin molecules in 3 endocytic patches from A vs. time. These and 21 other data sets were aligned on the same timescale by temporal superresolution alignment of the intensities to calculate the averaged numbers over time (black curve) ± 1 SD from the average (gray curves). Time zero is the time when the average number of fimbrin molecules peaked.
Mentions: We collected data on the time course of the accumulation and disappearance of the actin cross-linking protein fimbrin in endocytic actin patches to test our alignment method on a dynamic process in live cells. We imaged fission yeast cells expressing Fim1p-mEGFP at 25°C in EMM5S medium on five consecutive confocal sections spaced at 360 nm along the z-axis. These imaging conditions allowed us to track patches during most of their lifetimes and verify that their entire fluorescence signal was collected and did not overlap with other patches. We restricted our analysis to patches with fluorescence in only three consecutive slices, which we believe to be single isolated patches. We used five successive rounds of continuous alignment to align our data on the time course of the numbers of fimbrin molecules in well-separated actin patches (Figure 3, B and C).

Bottom Line: These methods allowed us to extract new information about endocytic actin patches in wild-type cells from measurements of the fluorescence of fimbrin-mEGFP.We show that the time course of actin assembly and disassembly varies <600 ms between patches.Our methods also show that the number of patches in fission yeast is proportional to cell length and that the variability in the repartition of patches between the tips of interphase cells has been underestimated.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology Department of Molecular Biophysics and Biochemistry Nanobiology Institute, Yale University, New Haven, CT 06520-8103 Institut Camille Jordan, UMR CNRS 5208, Université de Lyon, 69622 Villeurbanne-Cedex, France Centre de Génétique et de Physiologie Moléculaire et Cellulaire, UMR CNRS 5534, Université de Lyon, 69622 Villeurbanne-Cedex, France.

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Related in: MedlinePlus